PMC

Analysis of the interaction of CV-1 to CV-5, CNTF, CLC and IL-6 with sCNTFR and sIL-6R by co-precipitation. A, co-precipitation of CV-1 to CV-5, CNTF, CLC, and IL-6 by sCNTFR-Fc conjugated to protein A-agarose beads and detection by Myc-mAb. B, co-precipitation of CV-1 to CV-5, CNTF, CLC, and IL-6 by sIL-6R-Fc conjugated to protein A-agarose beads and detection by Myc-mAb. (+), input control of the cytokine. WB, Western blot.

Biological activity of CV-1 to CV-5, CNTF, CLC, IC-7, IL-6, and LIF on cells expressing gp130/LIFR and CNTFR/gp130/LIFR. A, Ba/F3-gp130-LIFR cell proliferation assay comparing the bioactivities of CNTF, IL-6, LIF, and CV-1 to CV-5 (1000–0.01 ng/ml). B, Ba/F3-CNTFR-gp130-LIFR cell proliferation assay comparing the bioactivities of CNTF, CLC, IL-6, LIF, IC-7, and CV-1 to CV-5 (1000–0.01 ng/ml). C, analysis of phosphorylation of STAT3 in Ba/F3-CNTFR-gp130-LIFR cells comparing the bioactivities of CNTF, CLC, IL-6, LIF, CV-1, and CV-5 (100 ng/ml).

Biological activity of CV-1 to CV-5, CNTF, CLC, IC-7, IL-6, and LIF on cells expressing IL-6R/gp130 and IL-6R/gp130/LIFR. A, Ba/F3-IL-6R-gp130 cell proliferation assay comparing the bioactivities of CNTF, IL-6, LIF, and CV-1 to CV-5 (1000–0.01 ng/ml). B, Ba/F3-IL-6R-gp130-LIFR cell proliferation assay comparing the bioactivities of CNTF, CLC, IL-6, LIF, IC-7, and CV-1 to CV-5 (1000–0.01 ng/ml). C, analysis of phosphorylation of STAT3 in HepG2 cells by Western blotting comparing the bioactivities of CNTF, CLC, IL-6, LIF, IC-7, and CV-1 to CV-5 (10 and 100 ng/ml). D, analysis of phosphorylation of STAT3 in HepG2 cells comparing the bioactivities of CV-1 to CV-5 (1000 ng/ml).

Pure CV-1 is monomeric and correctly folded. A, CV-1 was expressed in E. coli, purified via nickel-nitrilotriacetic acid agarose, and refolded. Finally, CV-1 was purified and analyzed by size-exclusion chromatography. CV-1 was eluted as a monomer. B, CD revealed the correct folding of CV-1. C, Coomassie stains of CV-1. CV-1 was separated by SDS-PAGE and visualized by Coomassie stain. D, Western blot analysis of purified CV-1 to CV5, CLC, and CNTF. Recombinant proteins were separated by SDS-PAGE and detected by Myc-mAb.

CNTF and CLC receptor-binding sites and schematic illustration of CNTF variants CV-1 to CV-5. A, illustration of CLC and CNTF binding site I to CNTFR/IL-6R, site II to gp130, and site III to LIFR. The minimal binding site Ia of CLC is highlighted in red, with the extended site Ia in red and orange. For CNTF, minimal site Ia is colored light blue, extended site Ib is colored light and dark blue. B, model of the CNTF·IL-6R (D2/D3) complex; Arg²⁸ is highlighted. C, schematic illustration of CNTF (light gray), CLC (dark gray), and CV-1 to CV-5. Site I from CLC is marked in orange and red; site I from CNTF is marked in light and dark blue. The exact mutations are shown on the right.